Bottom Line:
Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade.In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl.Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

ABSTRACTIn an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

Mentions:
Myristoylation has been shown to localize signaling proteins including Src family kinases to the membrane compartment and in many cases to be critical for their function (29). As SLAP-2 contains a putative NH2-terminal consensus motif (MGX1–4T/S) for addition of a myristoyl group, we generated an amino acid substitution in the myristoylation motif within a FLAG-tagged full length SLAP-2 (SLAP-2-myr) and examined its effect on antigen receptor–induced signaling. SLAP-2-myr in the pTRA-IRES.GFP vector was introduced into tTA-BJAB cells. The infected GFP-positive cells were sorted and assayed for anti-IgM–induced CD69 expression (Fig. 5 A) and SLAP-2 protein expression (Fig. 5 C). A ∼5-fold upregulation of CD69 expression was observed in vector-infected cells after BCR cross-linking (data not shown). Expression of wild-type SLAP-2 suppressed anti-IgM–induced CD69 levels by ∼2-fold (Fig. 5 A). In contrast, anti-IgM–induced CD69 expression in cells infected with SLAP-2-myr was comparable to the vector control (Fig. 5 A). Although SLAP-2-myr was expressed at slightly lower levels than wild-type SLAP-2 (Fig. 5 C), the complete loss of activity when the putative myristoylation site was disrupted suggests that this motif may play an important role in SLAP-2 function in BJAB cells. Wild-type SLAP-2 and SLAP-2-myr were also expressed in tTA-Jurkat cells at approximately equal levels (Fig. 5 D) and their effects on TCR signaling were examined (Fig. 5 B). TCR cross-linking stimulated a ∼4.5-fold upregulation of CD69 expression in vector-infected cells (data not shown). Expression of wild-type SLAP-2 strongly blocked upregulation of CD69 in response to TCR cross-linking (Fig. 5 B). Once again, mutation of the myristoylation consensus sequence significantly compromised this activity, suggesting that this motif is also important for SLAP-2 function in T lymphocytes.

Mentions:
Myristoylation has been shown to localize signaling proteins including Src family kinases to the membrane compartment and in many cases to be critical for their function (29). As SLAP-2 contains a putative NH2-terminal consensus motif (MGX1–4T/S) for addition of a myristoyl group, we generated an amino acid substitution in the myristoylation motif within a FLAG-tagged full length SLAP-2 (SLAP-2-myr) and examined its effect on antigen receptor–induced signaling. SLAP-2-myr in the pTRA-IRES.GFP vector was introduced into tTA-BJAB cells. The infected GFP-positive cells were sorted and assayed for anti-IgM–induced CD69 expression (Fig. 5 A) and SLAP-2 protein expression (Fig. 5 C). A ∼5-fold upregulation of CD69 expression was observed in vector-infected cells after BCR cross-linking (data not shown). Expression of wild-type SLAP-2 suppressed anti-IgM–induced CD69 levels by ∼2-fold (Fig. 5 A). In contrast, anti-IgM–induced CD69 expression in cells infected with SLAP-2-myr was comparable to the vector control (Fig. 5 A). Although SLAP-2-myr was expressed at slightly lower levels than wild-type SLAP-2 (Fig. 5 C), the complete loss of activity when the putative myristoylation site was disrupted suggests that this motif may play an important role in SLAP-2 function in BJAB cells. Wild-type SLAP-2 and SLAP-2-myr were also expressed in tTA-Jurkat cells at approximately equal levels (Fig. 5 D) and their effects on TCR signaling were examined (Fig. 5 B). TCR cross-linking stimulated a ∼4.5-fold upregulation of CD69 expression in vector-infected cells (data not shown). Expression of wild-type SLAP-2 strongly blocked upregulation of CD69 in response to TCR cross-linking (Fig. 5 B). Once again, mutation of the myristoylation consensus sequence significantly compromised this activity, suggesting that this motif is also important for SLAP-2 function in T lymphocytes.

Bottom Line:
Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade.In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl.Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

ABSTRACTIn an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.